U.S. patent application number 15/561350 was filed with the patent office on 2018-03-22 for composition for three-dimensional printing, method for preparing same, and method for manufacturing three-dimensional structure using same.
This patent application is currently assigned to POSTECH ACADEMY-INDUSTRY FOUNDATION. The applicant listed for this patent is POSTECH ACADEMY-INDUSTRY FOUNDATION, T&R BIOFAB CO., LTD.. Invention is credited to Dong-Woo CHO, Jin-Ah JANG.
Application Number | 20180078677 15/561350 |
Document ID | / |
Family ID | 56979141 |
Filed Date | 2018-03-22 |
United States Patent
Application |
20180078677 |
Kind Code |
A1 |
CHO; Dong-Woo ; et
al. |
March 22, 2018 |
COMPOSITION FOR THREE-DIMENSIONAL PRINTING, METHOD FOR PREPARING
SAME, AND METHOD FOR MANUFACTURING THREE-DIMENSIONAL STRUCTURE
USING SAME
Abstract
Provided is a three-dimensional printing composition including
decellularized extracellular matrix; and riboflavin as a
crosslinking agent. A three-dimensional structure having high
mechanical strength can be prepared by performing a printing
process using the three-dimensional printing composition according
to the present invention and a layer-by-layer process through
crosslinking under UVA light to prepare a three-dimensional
structure configuration; and then performing thermal gelation of
the three-dimensional structure configuration. Further provided is
a method for preparing said three-dimensional printing composition
and a method for preparing a three-dimensional structure using said
three-dimensional printing composition.
Inventors: |
CHO; Dong-Woo; (Seoul,
KR) ; JANG; Jin-Ah; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
POSTECH ACADEMY-INDUSTRY FOUNDATION
T&R BIOFAB CO., LTD. |
Pohang-si, Gyeongsangbuk-do
Siheung-si, Gyeonggi-do |
|
KR
KR |
|
|
Assignee: |
POSTECH ACADEMY-INDUSTRY
FOUNDATION
Pohang-si, Gyeongsangbuk-do
KR
T&R BIOFAB CO., LTD.
Siheung-si, Gyeonggi-do
KR
|
Family ID: |
56979141 |
Appl. No.: |
15/561350 |
Filed: |
February 25, 2016 |
PCT Filed: |
February 25, 2016 |
PCT NO: |
PCT/KR2016/001839 |
371 Date: |
September 25, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/3633 20130101;
A61L 2430/40 20130101; B33Y 70/00 20141201; A61L 27/227 20130101;
A61L 27/18 20130101; A61L 27/26 20130101; B33Y 10/00 20141201; A61L
27/3687 20130101 |
International
Class: |
A61L 27/36 20060101
A61L027/36; A61L 27/22 20060101 A61L027/22; A61L 27/26 20060101
A61L027/26; A61L 27/18 20060101 A61L027/18; B33Y 10/00 20060101
B33Y010/00; B33Y 70/00 20060101 B33Y070/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 2015 |
KR |
10-2015-0042412 |
Claims
1. A three-dimensional printing composition comprising a
decellularized extracellular matrix; and riboflavin as a
crosslinking agent.
2. The composition of claim 1, wherein the decellularized
extracellular matrix is obtained by decellularization of heart
tissues, cartilage tissues, bone tissues, adipose tissues, muscle
tissues, skin tissue, mucosal epithelial tissues, amnion tissues,
or corneal tissues which are externally discharged from the
body.
3. The composition of claim 1, wherein the decellularized
extracellular matrix is present in an amount ranging from 1 to 4%
by weight, based on the total weight of the composition.
4. The composition of claim 1, wherein the riboflavin is present in
an amount ranging from 0.001 to 0.1% by weight, based on the total
weight of the composition.
5. The composition of claim 1, further comprising one or more acids
selected from the group consisting of acetic acid and hydrochloric
acid; one or more proteinases selected from the group consisting of
pepsin and matrix metalloproteinase; and a pH adjusting agent.
6. The composition of claim 5, comprising 1 to 4% by weight of the
decellularized extracellular matrix; 0.001 to 0.1% by weight of the
riboflavin; 0.03 to 30% by weight of one or more acids selected
from the group consisting of acetic acid and hydrochloric acid; 0.1
to 0.4% by weight of one or more proteinases selected from the
group consisting of pepsin and matrix metalloproteinase; and a pH
adjusting agent, based on the total weight of the composition.
7. The composition of claim 5, wherein the viscosity at 1 s.sup.-1
shear rate when measured at 15.degree. C. ranges from 1 to 30
PaS.
8. A method for preparing a three-dimensional printing composition
comprising: (a) adding a decellularized extracellular matrix to one
or more acid solutions selected from the group consisting of acetic
acid and hydrochloric acid, (b) adding one or more proteinases
selected from the group consisting of pepsin and matrix
metalloproteinase to the solution obtained from Step (a), followed
by stirring the mixture to obtain a solution, and (c) adding
riboflavin and a pH adjusting agent to the solution obtained from
Step (b).
9. The method of claim 8, wherein the decellularized extracellular
matrix is obtained by decellularization of heart tissues, cartilage
tissues, bone tissues, adipose tissues, muscle tissues, skin
tissue, mucosal epithelial tissues, amnion tissues, or corneal
tissues which are externally discharged from the body.
10. The method of claim 8, wherein the decellularized extracellular
matrix is used in an amount ranging from 1 to 4% by weight, based
on the total weight of the composition.
11. The method of claim 8, wherein the riboflavin is used in an
amount ranging from 0.001 to 0.1% by weight, based on the total
weight of the composition.
12. A method for preparing a three-dimensional structure
comprising: (i) performing a printing process using the
three-dimensional printing composition of claim 1, and a
layer-by-layer process through crosslinking under UVA light, to
form a three-dimensional structure configuration; and (ii)
performing thermal gelation of the three-dimensional structure
configuration obtained from Step (i) at a temperature of 15.degree.
C. or more, to prepare a three-dimensional structure.
13. The method of claim 12, wherein the crosslinking in each
layer-by-layer process is performed for 1 to 10 minutes.
Description
TECHNICAL FIELD
[0001] The present invention relates to a three-dimensional
printing composition. And also, the present invention relates to a
method for preparing said three-dimensional printing composition
and a method for preparing a three-dimensional structure using said
three-dimensional printing composition.
BACKGROUND ART
[0002] Three-dimensional printing refers to fabricating a
complicated skeletal structure through converting the configuration
information derived from medical data of the tissues or organs
having complicated configurations to the G-codes and then
performing a layer-by-layer process using the same. Such a
three-dimensional printing is also referred to as
`three-dimensional bioprinting (3D bioprinting)`. For example, `the
multi-head tissue/organ printing system`, which is one of the
representative three-dimensional printing techniques, consists of
two pneumatic syringes for injecting materials by air pressure and
two piston syringes for injecting materials in a nano-liter unit
using a step motor, thereby being capable of utilizing various
materials at the same time. Typically, thermoplastic biocompatible
polymers, such as PLA (polylactic Acid), PGA (poly-glycolic acid),
PLGA (poly-lactic-co-glycolid acid), PCL (polycaprolactone), or a
mixture thereof, are loaded into the pneumatic syringes, so as to
prepare a three-dimensional structure. In addition, hydrogels,
including collagen, hyaluronic acid, gelatin, alginate, chitosan or
fibrin and the like, are loaded into the piston syringes, so as to
prepare a three-dimensional structure.
[0003] A crucial aspect of bioprinting is that the printing process
should be cytocompatible, as it requires the dispensing of
cell-containing media. This restriction reduces the choice of
materials because of the necessity to operate in an aqueous or
aqueous gel environment. Therefore, hydrogels using the materials
such as gelatin, gelatin/chitosan, gelatin/alginate,
gelatin/fibronectin, Lutrol F127/alginate, alginate and the like
are being used for preparing diverse tissues ranging from liver to
bone. Said hydrogels or mixtures of hydrogel and cells used for
bioprinting are also referred to as `bioink`.
[0004] Normally, cells remain located specifically in their
original deposited position during the whole culture period, as
they are unable to adhere or degrade the surrounding alginate gel
matrix (Fedorovich, N. E. et al. Tissue Eng. 13, 1905-1925 (2007)).
Thus, although there were some successful reports about bioprinting
of cell-printed structure, minimal cells-material interactions and
inferior tissue formation are the foremost concerns. Actually,
these materials cannot represent the complexity of natural
extracellular matrices (ECMs) and thus are inadequate to recreate a
microenvironment with cell-cell connections and three-dimensional
(3D) cellular organization that are typical of living tissues.
Consequently, the cells in those hydrogels cannot exhibit intrinsic
morphologies and functions of living tissues in vivo. It is thus
ideal if cells are provided the natural microenvironment similar to
their parent tissue. Decellularized extracellular matrix (dECM) is
the best choice for doing so, as no natural or man-made material
can recapitulate all the features of natural extracellular matrix.
Moreover, ECM of each tissue is unique in terms of composition and
topology, which is generated through dynamic and reciprocal
interactions between the resident cells and microenvironment.
Recent studies of cells and ECM isolated from tissues and organs
highlight the necessity of tissue specificity for preserving
selected cell functions and phenotype (Sellaro, T. L. et al. Tissue
Eng. Part A 16, 1075-1082 (2010); Petersen, T. H. et al. Science
329, 538-541 (2010); Uygun, B. E. et al. Nat. Med. 16, 814-821
(2010); Ott, H. C. et al. Nat. Med. 16, 927-933 (2010); Flynn, L.
E. Biomaterials 31, 4715-4724 (2010)). The dECM materials are
harvested and typically processed as two-dimensional (2D) scaffolds
from various tissues, including skin, small intestinal submucosa,
where at the initial stages the infiltrating or seeded cell
populations depend on diffusion of oxygen and nutrient for their
survival until a supporting vascular network develops. However,
printing tissue analogue structures requires a fabrication approach
to devise a highly open porous 3D structure to allow the flow of
nutrients. The present inventors have developed a three-dimensional
printing method for printing cell-laden constructs with the use of
dECM bioink capable of providing an optimized microenvironment
conducive to the growth of three-dimensional structured tissue. The
cell-laden constructs are able to reconstitute the intrinsic
cellular morphologies and functions (Falguni Pati, et al., Nat
Commun. 5, 3935 (2014)).
[0005] Meanwhile, the structure prepared by three-dimensional
printing with a dECM bioink should have a mechanical strength
capable of maintaining the three-dimensional configuration. For
example, in extrusion-based printing such as a multi-head
tissue/organ printing system, a three-dimensional structure
configuration is formed while the bioink in a pre-gel form is being
maintained at a temperature below about 15.degree. C. during the
extrusion from the syringe. The resulting three-dimensional
structure configuration is subject to thermal processing or
post-print crosslinking, as a process for imparting an appropriate
mechanical strength. The thermal processing is carried out for
example through gelation in a humid incubator at about 37.degree.
C. The post-print crosslinking is carried out through crosslinking
by treating the three-dimensional structure configuration with a
solution of crosslinking agent such as glutaraldehyde. However,
since the three-dimensional structure obtained through gelation by
the thermal processing shows relatively low mechanical strength, it
is difficult to manufacture the organ that requires satisfactory
mechanical strength. And also, post-print crosslinking requires the
use of toxic cross-linking agents, such as glutaraldehyde, which
causes a safety problem. In addition, the insufficient crosslinking
inside the three-dimensional structure results in the problem that
non-uniformly crosslinked three-dimensional structures are
obtained.
DISCLOSURE
Technical Problem
[0006] The present inventors carried out various studies in order
to develop an improved method for preparing a three-dimensional
structure having high mechanical strength by three-dimensional
printing processes. The present inventors have developed a method
capable of preparing a three-dimensional structure having high
mechanical strength uniformly, the method of which includes
performing a printing process using the three-dimensional printing
composition comprising riboflavin having a high safety as a
crosslinking agent and a layer-by-layer process through
crosslinking under UVA light to prepare a three-dimensional
structure configuration; and then performing thermal gelation of
the three-dimensional structure configuration. That is, the present
inventors have newly developed a crosslinking-thermal gelation
method which includes the use of riboflavin.
[0007] Therefore, it is an object of the present invention to
provide a three-dimensional printing composition comprising
riboflavin as a crosslinking agent.
[0008] It is another object of the present invention to provide a
method for preparing said three-dimensional printing
composition.
[0009] It is still another object of the present invention to
provide a method for preparing a three-dimensional structure using
said three-dimensional printing composition.
Technical Solution
[0010] In accordance with an aspect of the present invention, there
is provided a three-dimensional printing composition comprising a
decellularized extracellular matrix; and riboflavin as a
crosslinking agent.
[0011] In the three-dimensional printing composition of the present
invention, the decellularized extracellular matrix may be obtained
by decellularization of heart tissues, cartilage tissues, bone
tissues, adipose tissues, muscle tissues, skin tissue, mucosal
epithelial tissues, amnion tissues, or corneal tissues which are
externally discharged from the body; and may be present in an
amount ranging from 1 to 4% by weight, based on the total weight of
the composition. And also, the riboflavin may be present in an
amount ranging from 0.001 to 0.1% by weight, based on the total
weight of the composition.
[0012] The three-dimensional printing composition of the present
invention may further comprise one or more acids selected from the
group consisting of acetic acid and hydrochloric acid; one or more
proteinases selected from the group consisting of pepsin and matrix
metalloproteinase; and a pH adjusting agent. In an embodiment, the
three-dimensional printing composition of the present invention may
comprise 1 to 4% by weight of the decellularized extracellular
matrix; 0.001 to 0.1% by weight of the riboflavin; 0.03 to 30% by
weight of one or more acids selected from the group consisting of
acetic acid and hydrochloric acid; 0.1 to 0.4% by weight of one or
more proteinases selected from the group consisting of pepsin and
matrix metalloproteinase; and a pH adjusting agent, based on the
total weight of the composition. And also, in the three-dimensional
printing composition of the present invention, the viscosity at 1
s.sup.-1 shear rate when measured at 15.degree. C. may range from 1
to 30 PaS.
[0013] In accordance with another aspect of the present invention,
there is provided a method for preparing a three-dimensional
printing composition comprising: (a) adding a decellularized
extracellular matrix to one or more acid solutions selected from
the group consisting of acetic acid and hydrochloric acid, (b)
adding one or more proteinases selected from the group consisting
of pepsin and matrix metalloproteinase to the solution obtained
from Step (a), followed by stirring the mixture to obtain a
solution, and (c) adding riboflavin and a pH adjusting agent to the
solution obtained from Step (b).
[0014] In accordance with still another aspect of the present
invention, there is provided a method for preparing a
three-dimensional structure comprising: (i) performing a printing
process using the three-dimensional printing composition and a
layer-by-layer process through crosslinking under UVA light, to
form a three-dimensional structure configuration; and (ii)
performing thermal gelation of the three-dimensional structure
configuration obtained from Step (i) at a temperature of 15.degree.
C. or more, to prepare a three-dimensional structure.
[0015] In an embodiment, the crosslinking in each layer-by-layer
process may be performed for 1 to 10 minutes.
Advantageous Effects
[0016] It has been found by the present invention that a
three-dimensional structure having high mechanical strength can be
prepared by performing a printing process using the
three-dimensional printing composition comprising riboflavin and a
layer-by-layer process through crosslinking under UVA light to
prepare a three-dimensional structure configuration; and then
performing thermal gelation of the three-dimensional structure
configuration. That is, the present invention provides a
crosslinking-thermal gelation method including the use of
riboflavin, which makes it possible to prepare a three-dimensional
structure having high mechanical strength uniformly. And also, the
present invention includes the use of riboflavin having a high
safety as a crosslinking agent, thereby being capable of avoiding
the use of toxic crosslinking agents such as glutaraldehyde.
Accordingly, the present invention can be usefully applied to
fabricating tissue-engineering scaffolds, cell-based sensors,
drug/toxicity screening models and tissue or tumour models, through
three-dimensional printing.
DESCRIPTION OF DRAWINGS
[0017] FIGS. 1a and 1 b show the optical microscopic images (FIG.
1a) and histological images (FIG. 1b) of the decellularized
extracellular matrix (hdECM) derived from heart tissues.
[0018] FIG. 2 shows the configuration of the three-dimensional
structure prepared according to the present invention, with using a
PCL framework.
[0019] FIG. 3 shows the configuration of the three-dimensional
structure prepared according to the present invention, without
using a PCL framework.
BEST MODE
[0020] The present invention provides a three-dimensional printing
composition comprising a decellularized extracellular matrix; and
riboflavin as a crosslinking agent.
[0021] The decellularized extracellular matrix may be obtained by
decellularization of tissues discharged from mammals such as human,
pig, cow, rabbit, dog, goat, sheep, chicken, horse and the like.
The tissues are not particularly limited, and for example include
heart tissues, cartilage tissues, bone tissues, adipose tissues,
muscle tissues, skin tissue, mucosal epithelial tissues, amnion
tissues, or corneal tissues, preferably heart tissues, cartilage
tissues, or bone tissue, more preferably heart tissues, cartilage
tissues, or bone tissues derived from pigs. The decellularization
may be performed according to or with minor modifications to known
methods disclosed in for example Ott, H. C. et al. Nat. Med. 14,
213-221 (2008), Yang, Z. et al. Tissue Eng. Part C Methods 16,
865-876 (2010), and the like. Preferably, the decellularization may
be carried out according to the decellularization method previously
reported by the present inventors, i.e., according to the
decellularization method disclosed in Falguni Pati, et al., Nat
Commun. 5, 3935 (2014). The obtained decellularized extracellular
matrix is typically stored in a lyophilized powder form. The amount
of the decellularized extracellular matrix is not particularly
limited. For example, the decellularized extracellular matrix may
be used in an amount ranging from 0.001 to 0.1% by weight,
preferably from 2 to 3% by weigh, based on the total weight of the
composition.
[0022] It has been found by the present invention that a
three-dimensional structure having high mechanical strength
uniformly can be prepared by performing a printing process using
the three-dimensional printing composition comprising riboflavin
having a high safety and a layer-by-layer process through
crosslinking under UVA light to prepare a three-dimensional
structure configuration; and then performing thermal gelation of
the three-dimensional structure configuration. That is, the present
invention provides a crosslinking-thermal gelation method including
the use of riboflavin. Said riboflavin may be used in a sufficient
amount for crosslinking under UVA light. For example, the
riboflavin may be used in an amount ranging from 0.001 to 0.1% by
weight, preferably from 0.01 to 0.1% by weight, based on the total
weight of the composition.
[0023] It is preferable that the three-dimensional printing
composition of the present invention is in the form of a
visco-elastic homogeneous solution having a range of pH 6.5 to 7.5,
in order to provide efficient three-dimensional printing.
Therefore, the three-dimensional printing composition of the
present invention may further comprise one or more acids selected
from the group consisting of acetic acid and hydrochloric acid; one
or more proteinases selected from the group consisting of pepsin
and matrix metalloproteinase; and a pH adjusting agent for
controlling the pH to the range of 6.5 to 7.5 (for example, sodium
hydroxide), in an aqueous medium. The acid functions to dissolve a
decellularized extracellular matrix. Preferably, the acid may be
acetic acid, hydrochloric acid, and the like. More preferably, the
acid may be used in the form of a 0.01M-10M acetic acid solution
(for example, about 0.5M acetic acid solution) or in the form of a
0.01M-10M hydrochloric acid solution. The proteinase functions to
digest the telopeptide in a decellularized extracellular matrix.
Preferably, the proteinase may be pepsin, matrix metalloproteinase,
and the like. The amount of the proteinase depends on the amount of
a decellularized extracellular matrix. For example, the proteinase
may be used in a ratio of 5 to 30 mg, preferably 10 to 25 mg, with
respect to 100 mg of a decellularized extracellular matrix. The pH
adjusting agent functions to neutralize the acid used for
dissolving a decellularized extracellular matrix. For example,
sodium hydroxide as the pH adjusting agent may be used in an amount
sufficient to control the pH to pH 6.5 to 7.5, preferably about pH
7.
[0024] In an embodiment, the three-dimensional printing composition
of the present invention may comprise 1 to 4% by weight of the
decellularized extracellular matrix; 0.001 to 0.1% by weight of the
riboflavin; 0.03 to 30% by weight of one or more acids selected
from the group consisting of acetic acid and hydrochloric acid; 0.1
to 0.4% by weight of one or more proteinases selected from the
group consisting of pepsin and matrix metalloproteinase; and a pH
adjusting agent, based on the total weight of the composition. And
also, the three-dimensional printing composition of the present
invention is preferably in the visco-elastic form which shows lower
viscosity according to increasing the shear rate thereof. For
example, in the three-dimensional printing composition of the
present invention, the viscosity at 1 s.sup.-1 shear rate when
measured at 15.degree. C. is preferably from 1 to 30 PaS. The
viscosity may be adjusted by appropriately controlling the amount
of aqueous medium (e.g., water, distilled water, PBS, physiological
saline, etc.).
[0025] The present invention also provides a method for preparing
said three-dimensional printing composition. That is, the present
invention provides a method for preparing a three-dimensional
printing composition comprising: (a) adding a decellularized
extracellular matrix to one or more acid solutions selected from
the group consisting of acetic acid and hydrochloric acid, (b)
adding one or more proteinases selected from the group consisting
of pepsin and matrix metalloproteinase to the solution obtained
from Step (a), followed by stirring the mixture to obtain a
solution, and (c) adding riboflavin and a pH adjusting agent to the
solution obtained from Step (b).
[0026] In the method of the present invention, the acid,
decellularized extracellular matrix, riboflavin, proteinase, and pH
adjusting agent are as described above.
[0027] The acid solution of Step (a) may be e.g., a 0.01M-0.5M
acetic acid solution, preferably an about 0.5M acetic acid
solution. The stirring of Step (b) may be carried out until
achieving complete solubilization of the decellularized
extracellular matrix. The stirring of Step (b) may be carried out
typically for 24 to 48 hours, but not limited thereto. Step (c) is
carried out at about 15.degree. C. or less, preferably at a low
temperature ranging from about 4.degree. C. to about 10.degree. C.,
in order to avoid gelation. The resulting three-dimensional
printing composition is in the form of pH-adjusted pre-gel, which
is stored preferably at about 4.degree. C.
[0028] The present invention also provides a method for preparing a
three-dimensional structure comprising: (i) performing a printing
process using the three-dimensional printing composition and a
layer-by-layer process through crosslinking under UVA light, to
form a three-dimensional structure configuration; and (ii)
performing thermal gelation of the three-dimensional structure
configuration obtained from Step (i) at a temperature of 15.degree.
C. or more, to prepare a three-dimensional structure.
[0029] The printing of Step (i) may be carried out by using known
three-dimensional printing methods (e.g., a printing method with
`the multi-head tissue/organ printing system`), according to the
methods disclosed in Falguni Pati, et al., Nat Commun. 5, 3935
(2014) and the like. For example, the printing may be performed by
using two syringes of the multi-head tissue/organ printing system.
That is, a polycaprolactone (PCL) framework is loaded into the
syringe, followed by heating to about 80.degree. C. to melt the
polymer. Said three-dimensional printing composition in the form of
pre-gel is loaded into the other syringe, followed by maintaining
the temperature at about 15.degree. C. or less, preferably at about
4.degree. C. to 10.degree. C. For fabrication of the PCL framework,
pneumatic pressure is applied in the range from 400 to 650 kPa. The
composition in the form of pre-gel is dispensed by using a
plunger-based low-dosage dispensing system. In addition, the
printing may be also carried out by dispensing only the composition
in the form of pre-gel using a plunger-based low-dosage dispensing
system, without using a polycaprolactone framework.
[0030] The crosslinking under UVA light may be carried out by
irradiating UVA light having 315 to 400 nm of wavelength,
preferably having about 360 nm of wavelength, for 1 to 10 minutes,
preferably for about 3 minutes. By repeatedly performing said
printing and said crosslinking under UVA light, i.e., the
layer-by-layer process, a three-dimensional structure configuration
becomes formed.
[0031] Step (ii) is carried out by performing thermal gelation of
the three-dimensional structure configuration obtained from Step
(i) at a temperature of 15.degree. C. or more. The thermal gelation
may be performed by standing the three-dimensional structure
configuration at a humid incubator the temperature of which is
maintained preferably at 20 to 40.degree. C., more preferably at
about 37.degree. C., for 5 to 60 minutes, preferably for 20 to 30
minutes.
[0032] The present invention will be described in further detail
with reference to the following examples. These examples are for
illustrative purposes only and are not intended to limit the scope
of the present invention.
[0033] The decellularized extracellular matrix used in the
following examples was obtained by using porcine heart tissues,
according to the method disclosed in Falguni Pati, et al., Nat
Commun. 5, 3935 (2014), and hereinafter referred to as `hdECM`. The
obtained hdECM was finally lyophilized and stored in the freezer
until the use thereof. The optical microscopic image and the
histological image of the hdECM are as shown in FIGS. 1a and 1
b.
Example 1: Preparation of the Three-Dimensional Printing
Composition
[0034] Lyophilized hdECM was crushed into powder using a mortar and
pestle with the help of liquid nitrogen. The hdECM powder (330 mg)
was added to a solution of 0.5M acetic acid and then pepsin (33 mg)
(P7125, Sigma-Aldrich) was added thereto. The mixture was stirred
at room temperature for 48 h. While maintaining the temperature of
the resulting solution at 10.degree. C. or less, riboflavin (2 mg)
was added thereto. The pH of the resulting solution was adjusted to
about pH 7 with dropwise addition of cold (10.degree. C. or less)
10M NaOH solution. The obtained solution in the form of pre-gel was
stored in the refrigerator at about 4.degree. C.
Example 2: Preparation of the Three-Dimensional Structure
[0035] A three-dimensional structure was fabricated using the
three-dimensional printing composition obtained in Example 1,
according to the method disclosed in Falguni Pati, et al., Nat
Commun. 5, 3935 (2014). Specifically, the polycaprolactone (PCL)
framework was loaded into the syringe (first syringe) of the
multi-head tissue/organ printing system (Jin-Hyung Shim et al., J.
Micromech. Microeng. 22 085014 (2012)) and then heated to about
80.degree. C. to melt the polymer. The three-dimensional printing
composition in the form of pre-gel obtained in Example 1 was loaded
to the other syringe (second syringe) and then maintained at
temperatures below about 10.degree. C. Pneumatic pressure of about
600 kPa was applied to the first syringe to fabricate the thin PCL
framework of 120 .mu.m thickness having a line width of less than
about 100 .mu.m, with a gap of about 300 .mu.m. The contents in the
second syringe was dispensed over the PCL framework and then the
UVA light of about 360 nm was irradiated thereon for 3 minutes to
crosslink the composition.
[0036] Then, the dispensing processes of the contents in the second
syringe and then the layer-by-layer processes through the
crosslinking were repeatedly carried out to form a
three-dimensional structure configuration. The resulting
three-dimensional structure configuration was placed in a humid
incubator (the temperature thereof: about 37.degree. C.) and then
subject to thermal gelation by standing for 30 minutes to prepare a
three-dimensional structure. The resulting three-dimensional
structure has a thickness of about 300 to 400 .mu.m. An example of
the configuration is as shown in FIG. 2.
Example 3: Preparation of the Three-Dimensional Structure
[0037] A three-dimensional structure was fabricated according to
the same procedures as in Example 2, except that the PCL framework
was not used. That is, the three-dimensional printing composition
in the form of pre-gel obtained in Example 1 was loaded to the
syringe of the multi-head tissue/organ printing system (Jin-Hyung
Shim et al., J. Micromech. Microeng. 22 085014 (2012)) and then
maintained at temperatures below about 10.degree. C. Pneumatic
pressure of about 600 kPa was applied to the syringe so as to
dispense the contents therein and then the UVA light of about 360
nm was irradiated thereon for 3 minutes to crosslink the
composition. Then, the dispensing processes of the contents in the
second syringe and then the layer-by-layer processes through the
crosslinking were repeatedly carried out to form a
three-dimensional structure configuration. The resulting
three-dimensional structure configuration was placed in a humid
incubator (the temperature thereof: about 37.degree. C.) and then
subject to thermal gelation by standing for 30 minutes to prepare a
three-dimensional structure. The resulting three-dimensional
structure has a thickness of about 400 .mu.m. An example of the
configuration is as shown in FIG. 3.
Comparative Example
[0038] The solution in the form of pre-gel was prepared according
to the same procedures as in Example 1, except that riboflavin was
not used.
Experimental Example
[0039] The solution in the form of pre-gel obtained in Example 1
was subject to crosslinking by irradiating the UVA light of about
360 nm thereon for 3 minutes, placed in a humid incubator (the
temperature thereof: about 37.degree. C.), and then subject to
thermal gelation by standing for 30 minutes to form a hydrogel
(Hydrogel A). And also, the solution in the form of pre-gel
obtained in Comparative Example was placed in a humid incubator
(the temperature thereof: about 37.degree. C.) and then subject to
thermal gelation by standing for 30 minutes to form a hydrogel
(Hydrogel B). The complex modulus at frequency of 1 rad/s was
measured for each of the obtained hydrogels, and the results are
shown in Table 1 below.
TABLE-US-00001 TABLE 1 Modulus (n = 3, 1 rad/s) Hydrogel A 10.58
.+-. 3.4 kPa Hydrogel B 0.33 .+-. 0.13 kPa
[0040] As can be seen from the results in Table 1 above, the
hydrogel obtained according to the present invention exhibits 10.58
kPa of modulus at frequency of 1 rad/s, which shows at least about
30-fold improvement in strength by the crosslinking.
* * * * *